Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System

TY - JOUR

T1 - Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System

AU - Bat-Erdene, Undramaa

AU - Billingsley, John M.

AU - Turner, William C.

AU - Lichman, Benjamin R.

AU - Ippoliti, Francesca M.

AU - Garg, Neil K.

AU - O'Connor, Sarah E.

AU - Tang, Yi

N1 - Funding Information: This work is support by NIH R01AT010001 to Y.T. The plasmid used for expression of NoxE was a gift from Prof. James Bowie. © 2021 American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

PY - 2021/8/6

Y1 - 2021/8/6

N2 - Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD+-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD+ to NADH and a low ratio of NADP+ to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450, and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD+-dependent dehydrogenase to the one-pot mixture led to ∼1 g/L of nepetalactone, the active cat attractant in catnip.

AB - Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD+-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD+ to NADH and a low ratio of NADP+ to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450, and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD+-dependent dehydrogenase to the one-pot mixture led to ∼1 g/L of nepetalactone, the active cat attractant in catnip.

KW - biocatalysis

KW - cofactor regeneration

KW - enzyme cascade

KW - natural products

KW - synthetic biochemistry

UR - http://www.scopus.com/inward/record.url?scp=85112309661&partnerID=8YFLogxK

U2 - 10.1021/acscatal.1c02267

DO - 10.1021/acscatal.1c02267

M3 - Article

AN - SCOPUS:85112309661

SN - 2155-5435

VL - 11

SP - 9898

EP - 9903

JO - ACS Catalysis

JF - ACS Catalysis

IS - 15

ER -